Hi
i have a set of RNAseq data with unbalanced batch effect (see table below). Batch 1 was made of single indexed kit and sequenced at time 1, while batch 2 was made of dual indexed kit and sequenced independently from batch 1.
sample 
batch 
groups 

1 
Naive_Dmt3aKO_rep1 
2 
Naive_Dmt3aKO 
2 
Naive_Dmt3aKO_rep2 
2 
Naive_Dmt3aKO 
3 
Naive_Dmt3aKO_rep3 
2 
Naive_Dmt3aKO 
4 
Naive_WT_rep1 
2 
Naive_WT 
5 
Naive_WT_rep2 
2 
Naive_WT 
6 
Naive_WT_rep3 
2 
Naive_WT 
7 
Th17_Dmt3aKO_rep1 
2 
Th17_Dmt3aKO 
8 
Th17_Dmt3aKO_rep2 
2 
Th17_Dmt3aKO 
9 
Th17_Dmt3aKO_rep3 
1 
Th17_Dmt3aKO 
10 
Th17_WT_rep1 
2 
Th17_WT 
11 
Th17_WT_rep2 
2 
Th17_WT 
12 
Th17_WT_rep3 
1 
Th17_WT 
13 
Th1_Dmt3aKO_rep1 
2 
Th1_Dmt3aKO 
14 
Th1_Dmt3aKO_rep2 
2 
Th1_Dmt3aKO 
15 
Th1_Dmt3aKO_rep3 
1 
Th1_Dmt3aKO 
16 
Th1_WT_rep1 
2 
Th1_WT 
17 
Th1_WT_rep2 
2 
Th1_WT 
18 
Th1_WT_rep3 
1 
Th1_WT 
19 
Th2_Dmt3aKO_rep1 
2 
Th2_Dmt3aKO 
20 
Th2_Dmt3aKO_rep2 
2 
Th2_Dmt3aKO 
21 
Th2_Dmt3aKO_rep3 
1 
Th2_Dmt3aKO 
22 
Th2_WT_rep1 
2 
Th2_WT 
23 
Th2_WT_rep2 
2 
Th2_WT 
24 
Th2_WT_rep3 
1 
Th2_WT 
From my exploratory analysis, I noticed batch 1 samples and batch 2 samples are clustered independently from each other. https://ibb.co/meWjcz
Thus, on my DE analysis design, I used batch as covariant to evaluate the batch effect DE.
groups < relevel(groups, ref="Naive_KO") batch < relevel(batch,ref="1") design < model.matrix(~batch+groups, data=y$samples) y_filtered < estimateDisp(y_filtered,design) fit < glmQLFit(y_filtered, design, robust=T)
I found 24439 genes were differentially expressed btw batch 1 and batch 2.
#### batch effect batch_DE < glmQLFTest(fit, coef=2) FDR < p.adjust(batch_DE$table$PValue, 'fdr') sum(FDR < 0.05) # 24439
My questions:
1. since Naive has only batch 2 samples no batch 1 samples, Can 24439 batch DE genes be caused by difference between other Th and Naive? In the linear regression, ~batch+groups
, we assume batch and group are independent. theoretically, those 24439 genes should be independent of group difference, but this unbalanced design really bothers me.
2. Will this unbalance design affect differential analysis between groups? for example, comparing Th1_WT to Naive_WT.